Nearly all licensed vaccines protect through antibodies rather than cell-mediated immunity. A critical aspect of vaccine-induced serological protection is the duration of antibody titer post-boost. Our basic understanding of T-cell dependent antibody responses involves presentation of antigens by dendritic cells to specific CD4+ T cells, which then proliferate and differentiate into helper T cells (Tfh) that engage antigen-engaged B cells that have moved to the T-B border of secondary lymphoid tissues (spleen or lymph node). These interactions can generate a population of short-lived, high-rate antibody secreting cells (the extrafollicluar antibody response), which contributes to acute host defense but not to long-lived protection. The latter involves migration of antigen-activated T cells and the associated antigen-specific B cells into the B cell follicle where they set up the germinal center reaction. Here continued T-B interaction leads to somatic hypermutation and isotype class switching, producing antibodies of higher affinity and different effector class, while generating both memory B cells and also plasmablasts that can become long-lived plasma cells if they reach the proper niche, which is mainly located in the bone marrow. While this general outline is well-established, the molecular signals that guide the engagement of T and B cells to generate a maximally productive response, the role of Tfh in determining the choice between memory B cells and long-lived plasma cells, and what determines how plasmablasts become long-lived plasma cells remain unclear. This project is a collaborative effort among laboratories with expertise in vaccine development, adjuvant function, and the cellular immune reactions that aims to examine how adjuvants affect each of these key steps in humoral immune responses and how variations in the quality and quantity of Tfh, stimuli for B cells, and niche space for plasma cells affects the peak titer and persistence of antibody responses post-vaccination. In FY14, progress has been made in systematically testing a variety of adjuvants with candidate antigens selected for a transmission-blocking malarial vaccine with respect to their capacity to produce durable antibody responses of adequate titer. Distinct adjuvants produced substantially different peak post-boost levels of Pfs25-specific antibodies, with a role also found the carrier protein. Surprisingly and in contrast to data from NHP studies performed previously, the decline in antibody titer over nearly a year showed a similar slope for all conditions. This raises the important question of whether this is a species-related difference or if it reflects the fact that the mice are clean in comparison to the NHP, influencing the availability of bone marrow niche space for long-lived plasma cells. This issue is being addressed in mouse experiments employing a new method for testing the bone marrow homing capacity of recently generated plasmablasts and their longevity in that compartment. Studies are now under way in conventionally reared animals and in those given strong infectious challenges to fill up the bone marrow plasma cell niche. We have also initiated studies examining the role of inflammatory signals in mobilizing bone marrow resident plasma cells and allowing their replacement with newly generated plasmablasts derived from vaccination. Additional experiments are examining whether the physiological state of the plasmablasts also controls the duration of the serological response, with preliminary studies showing that we can detect differential rates of titer decline in mice given plasmablasts from different priming regimens. To complement these functional studies and directly examine the plasma cell niche in bone marrow, we have developed new methods for long bone whole mount microscopy that allows detection of antigen specific and well as IgG producing plasma cells and the detailed characterization of the niche in which they reside. In prior work, we used a model system in which mice were immunized with a peptide epitope from Toxoplasma emulsified in Complete Freunds Adjuvant and then measured T cell responses using a specific Class II MHC tetramer at day 14 post-injection. With this protocol we found that approximately 7% of the CD4+, Foxp3- cells in draining lymph nodes from immunized mice were Tfh as defined by co-staining with CXCR5 and BCL-6; of these roughly 15% were tetramer positive. A more stringent analysis using a combination of 4 Tfh canonical markers revealed that approximately 1% of the CD4+, Foxp3- cells in immunized mice were CXCR5+, BCL-6 +, ICOShi, PD1hi versus 0.1 % in sham immunized mice. These responses were substantially reduced in immunized mice deficient in the TLR/IL-1R adaptor MyD88. This work is currently being extended with kinetic analyses and to studies with Incomplete Freunds adjuvant to evaluate the contribution of the mycobacterial component in CFA to Tfh induction. Additional studies are underway using a new set of Pfs25-protein conjugates to allow more definitive tracking of Tfh responses using tetramers. Pfs25 has been conjugated with the mycobacterial antigen Ag85b, for which the specific peptide determinant is well characterized. This will also allow imaging studies of the dynamics of T-DC and T-B interactions during development of the humoral response using TCR transgenic P25 T cells specific for Ag85b. This will permit direct and quantitative analysis of Tfh formation via dendritic cell and antigen-specific B cell interactions, assessment of B cell activation by antigen and innate stimuli, examination of the outcome of Tfh-B cell interactions in germinal centers, and determination of the proportion of memory B cells vs. plasmablasts formed by this latter response. The data generated by these rodent experiments will be used to plan NHP studies focused on determining if the top candidate adjuvants produce the same types of responses in these primates at the macro (i.e., antibody titer) level and with respect to the cellular events involved, such as bone marrow niche regulation. A key aim of this project is the identification of vaccine formulations that will extend the duration of the antibody response against malaria vaccine candidates. Our prototype target malaria antigen is Pfs25, which is undergoing Phase 1 trials in humans as a Pichia-expressed recombinant protein conjugated to the carrier protein ExoProtein A (EPA) expressed in E. coli with a molar ratio of 3:1, and formulated with the commercially available adjuvant Alhydrogel: Pfs25-EPA/Alhydrogel. Pfs25-EPA/Alhydrogel is undergoing Phase 1 trials in malaria-nave volunteers in the US (dose-escalating trial) and malaria-experienced volunteers in Mali (dose-escalating; double-blinded; placebo-controlled trial). Sera collected from volunteers in the US and Mali are being assessed for seroreactivity by standardized ELISA, and transmission-blocking antibody activity is measured in membrane feeding assays. Serum antibody levels in either assay are being measured before and after each vaccine dose, and then periodically thereafter to assess the duration of the antibody response, including the functional antibody response. In Mali, mosquitoes are fed directly on vaccinees to determine their infectivity/malaria transmission potential, and this will be related to the antibody measurements. The Pfs25-EPA/Alhydrogel product will be a benchmark against which we will compare novel Pfs25 products and formulations in our animal studies. Our animal studies of adjuvants to date support our plan to test Pfs25-based conjugate vaccines using alternative adjuvants in humans, and our initial focus is on the commercial product AS01 from GSK and a similar product (GLA + QS21 in liposomal formulation) from IDRI that has not yet been in the clinic.